ES2953463T3 - Bioelectronic pharmaceuticals - Google Patents

Abstract

Disclosed are methods of treating a subject having a disease or disorder comprising providing the subject with an acoustic energy stimulus derived from a disease-specific, condition-specific, endogenous mediator-specific, or pharmacological agent-specific neurogram in an amount and effective way to treat the disease or disorder. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Bioelectronic pharmaceuticals

Government Funding Statement

This invention was made with government funding under grant number W911NF-09-1-0125 awarded by the Defense Advanced Research Projects Agency (DARPA). The government has certain rights over the invention.

Background of the invention

Throughout this application reference is made in parentheses to various publications. Full citations to these references can be found at the end of the specification.

The nervous system developed over evolutionary time to optimize survival in response to signals from the internal and external environment. In mammals, chemical, mechanical, and electromagnetic signals are detected by neurons, which propagate action potentials to the central nervous system (CNS). These comprise the afferent arcs of the reflex circuits that maintain homeostasis of the body. This fundamental principle of detecting changes in the environment to mount appropriate reflex responses is fundamental to the physiological mechanisms that allow not only homeostasis but also the adaptability and survival of species.

Thirty years ago, it was discovered that products of the immune system, including cytokines and other mediators, could be perceived by the nervous system, prompting the suggestion that the immune system could serve as a functional sensory modality (1). In this context, foreign invaders, microbial products, and other exogenous immune stimulators culminate in the release of cytokines. These immune products can, in turn, interact with the peripheral nervous system and CNS to trigger neurophysiological responses; However, the question remains whether sensory neuronal signals are encoded in specific cytokine patterns.

There has been an expanding body of knowledge outlining the extensive interface between the nervous and immune systems. Similar to the neuronal control of general physiological and metabolic states of the body, systemic inflammatory pathways can be modulated by the CNS, with the archetypal pathway being the inflammatory reflex of the vagus nerve (VN) (2). In its efferent arc, electrical signals travel down the vagus nerve to the celiac ganglion from where the splenic nerve further propagates the signal to the spleen. Within the spleen, a specialized subset of T lymphocytes completes the link between the nervous and immune systems (3, 4). Acetylcholine, which is released by these T cells, downregulates cytokine production by resident macrophages, resulting in a systemic anti-inflammatory effect (3).

In contrast to the well-mapped motor arc, the afferent arc remains incompletely understood. Notably, the vagus nerve is primarily sensory, such that numerous afferent signals related to physiological state travel along the vagus nerve from the periphery to the CNS. The notion that these signals could include the inflammatory state of the animal is often neglected. The pioneering work of Niijima and collaborators (5-7) led them to postulate that IL-1p could activate peripheral afferents from the vagus nerve that would signal the presence of this cytokine to the CNS. Physiological studies have shown that an intact vagus nerve is required for a pyrexia response to intra-abdominal administration of IL-1p, further corroborating the notion that the vagus nerve could be a primary sensor of peripheral inflammation for the CNS (8, 9). Parallel studies in isolated sensory neurons show that neurons express a variety of cytokine receptors, such as TNF and IL-1p receptors, and are able to change their activation thresholds when exposed to the corresponding exogenous cytokines (10-12 ). Steinberg et al "Pro- and Anti-inflammatory Cytokines Differentially Activate the Sensory Vagus Nerve" (American Society of Anesthesiologists, Abstract, October 14, 2014, page 1, XP055482730) discloses intraperitoneal administration of proinflammatory tumor necrosis factor (TNF) purified or interleukin-1 (IL-1 •) that leads to an increase in electrical activity in the cervical vagus nerve while IL-10 does not. Combined, these studies suggest that the vagus nerve is an important substrate for a peripheral neural network capable of capturing real-time signals related to changes in peripheral inflammatory and immune states. The publication "Spike2 for Windows" (Cambridge Electronics Design Limited, January 1, 2017 (2017 01-01), XP055519070 is a user manual for signal data acquisition and analysis software.

The present invention addresses the need for improved treatments of diseases and disorders, in particular that do not require the administration of drugs to a subject. The methods disclosed herein use a stimulus pattern derived from a disease-specific, condition-specific, endogenous mediator-specific, or pharmacological agent-specific neurogram of the vagus nerve to produce an acoustic stimulus pattern that can be used subsequently to treat the disease or disorder.

Summary of the invention

The present invention provides methods for producing a .wav file wherein the .wav file can be played as an acoustic energy stimulus for later use in the treatment of a subject having a disease or disorder, wherein the disease or disorder is hyperglycemia or hypoglycemia, the method comprising generating the .wav file based on a neurogram specific to an endogenous mediator or specific to a pharmacological agent according to claim 1.

In particular, the invention provides a method of producing a .wav file wherein the .wav file can be played as an acoustic energy stimulus for subsequent use in monitoring blood glucose levels in subjects comprising generating the file .wav based on a hypoglycemia- or hyperglycemia-specific vagus nerve neurogram according to claim 1. The aspects described herein below as "not currently claimed" do not form part of the claimed invention, but are useful for understand the claimed invention, which is defined by the independent claims.

Brief description of the drawings

Fig. 1A-1D. Recording of compound action potentials from the vagus nerve. (A) Simultaneous recordings (2 of 3 electrodes shown) of spontaneous activity in the vagus nerve (VN). (B) Left, excitation of the vagus nerve by KCl (4 mM, gray box) applied to the surgical field. Right, Top, period of quiescent activity before KCl (marked by i). Middle part, period of intense peaks during KCl (marked by ii). The line indicates the adaptive threshold used to detect compound action potentials (CAPs). Bottom, the identified CAPs are aligned with the previous trace. (C) Inset, evoked CAPs are elicited by brief electrical pulses that stimulate the vagus nerve. The graph shows the CAP area (mean ± SD), which becomes larger with increasing stimulation intensity (pre circles). Lidocaine (2%) largely blocks evoked CAPs (post circles). (D) Inset, CAP elicited prior to treatment with tetrodotoxin (100 μM). The graph shows the area of CAP (mean ± SD) that is completely blocked by the drug. Scale bars (x, y), A, 100 ms, 100 pV; B left, 1 s, 20 pV; B right, 10 ms, 20 pV; C, D, 2 ms, 40 pV.

Fig. 2A-2E. Vagus nerve afferent fibers carry TNF-induced neurograms; aspect not currently claimed but considered useful to understand the invention. (A) Upper part, trace showing the spontaneous activity of the cervical vagus nerve. Bottom, trace showing cervical vagus nerve activity after peripheral injection of TNF (50 μg dose, marked with an arrow). (B) Graph depicting CAP firing frequency for control and TNF-induced neurograms (shown in A). The 10-minute period immediately after TNF injection is used to calculate the CAP discharge rate, as well as the equivalent period from baseline. (C) Diagrams of the surgical vagotomies used to test the direction of TNF-induced neurogram flow. A proximal transection (Prox.), between the electrodes and the brain, isolates the afferent component, while a distal transection isolates the efferent branch. (D) Graph showing CAP discharge frequency at 60-s intervals (mean ± SEM, line ± shaded area) starting 10 min before TNF injection (50 μg dose) at time zero. Data represent N = 3 for each of the proximal (filled circles) and distal (open circles) vagotomies. (E) Graph showing CAP frequencies (mean ± SEM) for the 10-min periods before and just after TNF in individual mice. Distal transection completely abolishes the effect of TNF, which is not affected by proximal transection (P = 0.03 for post values, t test), indicating that afferent fibers are required.

Fig. 3A-3E. Time course for TNF- and IL-1p-mediated neurograms; aspect not claimed currently considered useful for understanding the invention. (A) Top, trace showing vagus nerve activity with vehicle (200 μl sterile saline, marked with an arrow), used as a control. Bottom, graph depicting CAP discharge frequency (mean ± SEM, gray area) for N = 5 mice, starting 10 min before saline injection. (B) Top, representative neurogram for TNF (50 μg dose, marked with an arrow). Bottom, graph showing mean frequency ± SEM (blue area) for N = 6 mice. (C) Top, representative neurogram for IL-1p (350 ng dose, marked with an arrow). Bottom, graph showing mean frequency ± SEM (green area) for N = 8 mice. (D) Graph depicting CAP discharge frequency (mean ± SEM) for TNF at low dose (5 μg, N = 6) and high dose (50 μg, N = 6). 'Pre' refers to the 30 second interval just before the injection and 'Post' refers to the 30 second interval 5 minutes after the injection. (E) Graph depicting CAP firing frequency (mean ± SEM) for IL-1p at low dose (35 ng, N = 8) and high dose (350 ng, N = 8). 'Pre' refers to the 30 second period just before the injection and 'Post' refers to the 30 second period 2.5 minutes after the injection. Fig. 4A-4C. Time-domain analysis of TNF- and IL-1p-mediated neurograms; aspect not currently claimed but considered useful to understand the invention. (A) Boxplot showing the difference (postinjection minus preinjection) in mean CAP frequency. For TNF (N = 6) and saline (N = 5), 10-minute intervals were used. For IL-1p (N = 8), 5-minute intervals were used. (B) Boxplot showing the difference in maximum CAP frequency. (C) Left, graph representing latency (mean ± SEM) for TNF and IL-1p responses. The former is slightly slower than the latter (P = 0.043, t test). Middle part, graph showing the duration (mean ± SEM) of neurograms induced by TNF and IL-1p. Right, graph presenting CAP frequency (mean ± SEM) of TNF and IL-1p responses.

Fig. 5A-5B. Ablation of cytokine-mediated neurograms in TNFR2- and IL-1R-deficient mice; aspect not currently claimed but considered useful to understand the invention. (A) Left, dashes representative 5 minutes after application of TNF (dose of 50 μg) in TNFR2 KO and wild-type mice. Right, graph showing individual CAP frequencies immediately before and after TNF (10 minute intervals) in TNFR2 KO animals (N = 3) and wild-type animals (N = 6) (B) Left, representative traces 150 seconds after IL-1p application (350 ng dose) in wild-type and IL-1R KO mice. Right, graph showing individual CAP frequencies immediately before and after IL-1p (5-min intervals) in IL-1R KO (N = 3) and wild-type (N = 8) animals. Fig. 6A-6B. Spectral analysis of neurograms mediated by TNF and IL-1p; aspect not currently claimed but considered useful to understand the invention. (A) Representative power spectral densities (PSDs) for TNF and IL-1p responses in unfiltered neurogram recordings. (B) Areas under the PSDs (range 20–400 Hz) were calculated for TNF and IL-1p. The area calculated for each response is shown for TNF and IL-1p, along with group averages (lines). Responses are statistically different (N = 7 for each group; P = 0.05, D = 0.71, Kolmogorov-Smirnov test), suggesting a potential biological substrate for cytokine discrimination within the CNS.

Fig. 7. Paradigm for inducing the inflammatory phenotype in naïve mice by acoustic stimulation using an IL1p-specific signal; aspect not currently claimed but considered useful to understand the invention. A reference or IL1p-specific signal is extracted from a vagus nerve neurogram obtained from mouse A and converted to a 'wav' file. The baseline or IL1p-specific signal was transferred to another naïve recipient B mouse by acoustic stimulation. Circulating cytokine levels were analyzed in mouse B after 1 h.

Fig. 8. Acoustic stimulation using IL1p-mediated neurogram induced increased levels of circulating inflammatory cytokines; aspect not currently claimed but considered useful to understand the invention. Naïve animals receive acoustic stimulation using a baseline or IL1p-mediated neurogram signal (1X or 3X serial activation). IL1p-specific but not baseline signal induced inflammatory phenotype in naïve recipient mice.

Fig. 9. Recording of hypoglycemic neurogram of the vagus nerve. Naïve mice received a bolus injection of insulin (6 mg/kg) and vagus nerve activity was recorded over time. Blood glucose levels were monitored every 2.5 minutes. Insulin induced a hypoglycemic state in mice, as illustrated by the top line in the figure, which indicates blood glucose levels in mg/dL.

Fig. 10. Euglycemic neurogram recording of the vagus nerve. Naïve mice received a bolus injection of glucagon (1 mg/kg) and vagus nerve activity was recorded over time. Blood glucose levels were monitored every 2.5 minutes. Blood glucose levels are indicated on the top line of the figure in mg/dl.

Fig. 11. Hyperglycemic neurogram recording of the vagus nerve. Naïve mice received a bolus injection of glucose, and vagus nerve activity was recorded over time. Blood glucose levels were monitored every 2.5 minutes. Blood glucose levels are indicated on the top line of the figure in mg/dl.

Fig. 12. Paradigm for increasing blood glucose levels in healthy human subjects by acoustic stimulation using a hypoglycemia-specific signal. A reference or hypoglycemia-specific signal was extracted from a vagus nerve neurogram obtained from mouse A. The signal was converted to a 'wav' file and transferred as an audio output to healthy human subjects. Blood glucose levels were monitored in human subjects at regular time intervals.

Fig. 13. Acoustic playback of the hypoglycemia-specific neurogram induced hyperglycemia in healthy human subjects. Neurograms of the vagus nerve were recorded from mice that received a bolus injection of insulin. Blood glucose levels decreased in mice given insulin. The neurogram was then played back as a 'wav' file for healthy human subjects and blood glucose levels were monitored over time (n=5/group).

Fig. 14. Acoustic playback of the hyperglycemia-specific neurogram induced hypoglycemia in healthy human subjects. Neurograms of the vagus nerve were recorded from mice that received a bolus injection of glucose. Blood glucose levels increased in the mice given glucose. The neurogram was then played back as a 'wav' file for healthy human subjects and blood glucose levels were monitored over time (n=3/group).

Fig. 15. Acoustic playback of the euglycemia-specific neurogram did not induce a change in blood glucose levels in healthy human subjects. Neurograms of the vagus nerve were recorded from mice that received a bolus injection of glucagon. Blood glucose levels did not change in mice given glucagon. The neurogram was then played back as a 'wav' file for healthy human subjects and blood glucose levels were monitored over time (n=5/group).

Fig. 16. Hydrocortisone-induced neurogram recording of a mouse vagus nerve. Naïve mice received a bolus injection of hydrocortisone (cortisol) (10 mg/mouse) and vagus nerve activity was recorded over time.

Fig. 17. Administration of cortisol-induced hypoglycemia in mice. Naïve mice received saline or cortisol via intraperitoneal administration. Blood glucose levels were monitored over time. Administration of cortisol but not saline induced hypoglycemia. Fig. 18. Acoustic playback of the cortisol-specific vagus neurogram in induced hyperglycemia in healthy human subjects. Neurograms of the vagus nerve were recorded from mice that received a bolus injection of hydrocortisone. A "coded signal control" was generated by transforming the signal specific cortisol in a signal encoded using a matlab code that generates a randomized signal with the same amplitudes, but random frequencies, maintaining the total power of the signal. The cortisol-specific neurogram or scrambled signal was then played back as a 'wav' file to healthy human subjects and blood glucose levels were monitored over time. (n=8/group).

Detailed description of the invention

The present invention provides a method of producing a .wav file wherein the .wav file can be played as an acoustic energy stimulus for later use in the treatment of a subject having a disease or disorder comprising generating the .wav file based on a neurogram specific to an endogenous mediator or specific to a pharmacological agent.

The disease or disorder according to the invention may be, for example, one or more of hyperglycemia or hypoglycemia. It can treat type 1 diabetes or type 2 diabetes, glucose intolerance. Another disease or disorder not currently claimed, may be, for example, inflammation, metabolic syndrome, trauma, bleeding, hemorrhagic shock, ischemia-reperfusion injury, nausea, vomiting, cancer, prostate cancer, arthritis, rheumatoid arthritis, colitis, sepsis , endotoxemia, colitis, pancreatitis, inflammatory bowel disease, Crohn's disease, fever, anorexia, pain, swelling, a host response to infection, an immune response, and a disease or disorder in which increased activity or level is desirable of a cytokine.

An acoustic energy stimulus derived from a cytokine-specific neurogram can subsequently be used to modulate a cytokine-specific physiological effect; This aspect is not currently claimed. According to the invention, a corrective stimulus pattern derived from a subject's glucose or insulin level can subsequently be used to control glucose and, therefore, can preferentially replace the administration of insulin to the subject.

The present invention provides for monitoring blood glucose levels in a subject by producing a .wav file that can be played as an acoustic energy stimulus, the .wav file being derived from a hypoglycemia- or hyperglycemia-specific neurogram recorded from a vagus nerve, thus controlling blood glucose levels in a subject.

In one embodiment provided for increasing blood glucose levels in a subject, the .wav file may be played as an acoustic energy stimulus, derived from a hypoglycemia-specific, insulin-specific, or cortisol-specific neurogram recorded from a vagus nerve. , thus increasing blood glucose levels in a subject. The hypoglycemia-specific neurogram can be recorded from a vagus nerve of a subject after, for example, administration of insulin or cortisol to the subject. The subject receiving the acoustic energy stimulus may have, for example, one or more of hypoglycemia, kidney failure, liver disease, and hypothyroidism.

In another embodiment provided for lowering blood glucose levels in a subject, the .wav file may be played as an acoustic energy stimulus, derived from a hyperglycemia-specific or glucose-specific neurogram recorded from a vagus nerve, thereby decreasing blood glucose levels. blood glucose levels in a subject. The hyperglycemia-specific neurogram may be recorded from a vagus nerve of a subject after, for example, administration of glucose to the subject. The subject receiving the acoustic energy stimulus may have, for example, one or more of type 1 diabetes mellitus, type 2 diabetes mellitus, metabolic syndrome, insulin resistance, glucose intolerance and hyperglycemia.

Electrical sampling of neuronal activity ("neurograms") can be obtained in human or non-human subjects, such as an animal model of a disease or disorder, where neurograms are disease-specific, condition-specific, neurograms. of an endogenous mediator or specific to a pharmacological agent.

The neurogram can be obtained, for example, in response to the administration of substances of physiological origin to a subject. For example, in response to the administration of a cytokine; This aspect is not currently claimed. The cytokine may be, for example, a chemokine, a colony-stimulating factor, high mobility group protein B1 (HMGB1), an interferon (IFN), an interleukin (for example, any of IL-1 to IL-36) , a lymphokine, macrophage migration inhibitory factor (MIF), a monokine, a transforming growth factor beta ( for example, TGF-p1, TGF-p2 and TGF-p3), a tumor necrosis factor (for example, TNFα or TNFp). The neurogram can be obtained, according to the invention in response to the administration of glucagon, glucose or insulin, or in response to a change in glucose levels, for example, blood glucose levels. The neurogram may, for example, be recorded in response to a pro-inflammatory signal or an anti-inflammatory signal; This aspect is not currently claimed.

The neurogram is obtained from the vagus nerve.

Electrical sampling of vagal neuronal activity ("neurograms") can be obtained from an animal model or from a human patient and is preferably obtained from a cervical vagus nerve.

Nerve recording can be carried out using, for example, an implantable electrode, such as, for example, a cuff-style electrode. Neurograms can be obtained from the same subject being subsequently treated with acoustic stimulation or from a different subject. The subject that is subsequently treated may be a human subject or a veterinary subject.

The acoustic energy stimulus may subsequently be applied to one or both αdos of the subject, including, for example, using headphones. Acoustic energy stimulation may include auditory and vibrational energy stimulation.

A hypoglycemia- or hyperglycemia-specific vagus nerve neurogram can subsequently be used to provide a subject with an acoustic energy stimulus in an amount and in a manner effective to treat hypoglycemia or hyperglycemia.

This invention will be better understood from the Experimental Details, set forth below. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as more fully described in the claims that follow.

EXPERIMENTAL DETAILS

EXAMPLE 1. Cytokine-specific neurograms recorded from the afferent vagus nerve Overview. Cytokine-related embodiments of Example 1 are not currently claimed, but are useful in understanding the invention.

Neural networks in the periphery send signals about the physiological state of the body to the central nervous system (CNS) through afferent fibers, and particularly through the vagus nerve (VN). As part of this network, the sensory vagus nerve responds to interleukin-1p (IL-1p) and lipopolysaccharide, and transmits these signals to the nucleus tractus solitarius within the CNS. The present study mapped peripheral neuronal activity that occurred in response to tumor necrosis factor (TNF) and IL-1p recording from the cervical vagus nerve of adult mice. Using selective surgical vagotomies, cytokine-generated neurograms were shown to be carried by afferent fibers of the vagus nerve. Low doses of cytokines (5 μg TNF or 35 μg IL-1p) did not improve baseline vagus nerve activity, while higher doses (50 μg TNF or 350 ng IL-1(3) triggered significant improvements. Analysis of the temporal dynamics and power spectral characteristics of TNF- and IL-1p-mediated neurograms revealed selective cytokine signals in the vagus nerve.

Materials and methods

Animals. All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the Feinstein Institute for Medical Research, Northwell Health (formerly the North Shore-LIJ Health System), which follows NIH guidelines for ethical treatment. of the animals. The studies used male BALB/c mice (8-12 weeks, weighing 20-30 g), which were purchased from The Jackson Laboratory (Bar Harbor, ME). They were housed at 25°C, with food and water ad libitum, and acclimated to a 12-h light-dark cycle for >3 days before conducting experiments.

Chemical products. Lidocaine, tetrodotoxin (Sigma-Aldrich), and human IL-1p (eBiosciences) were purchased. Recombinant, label-free human TNF was produced in-house. After expression in E. coli, TNF was purified using a cation exchange column and endotoxins were removed by phase separation with Triton X-114.

In vivo NV recordings. Mice were fasted (3-4 h) before each experiment. Each was induced (2.5% isoflurane) and maintained (1.5% isoflurane) in the supine position for surgery. A midline cervical incision was made and the left cervical branch of the vagus nerve was isolated from the accompanying carotid bundle. Under magnification, the connective tissue was mechanically removed and the vagus nerve was placed over three custom-made silver hook electrodes. Electrophysiological signals were digitized (sampling rate, 32 kHz) through a data acquisition system (Digital Lynx 4SX, Cheetah v5 software, Neuralynx, Bozeman, MT) and referenced to an animal ground electrode placed between the skin and the right salivary gland. In all experiments, the vagus nerve was protected from desiccation and isolated by bathing the surgical field with mineral oil. After acquisition of baseline activity (15-20 min), animals were injected ip with TNF, IL-1p, or saline control and recordings continued for 20-30 min after injection. The experimenter was grounded whenever he handled the animal during the recordings.

Vagotomies. A surgical transection of the vagus nerve was completed proximal or distal to the hook electrodes, considering the brain as a reference point (Fig. 2D). After placement of the vagus nerve on the hook electrodes, a silk suture was passed underneath and secured to the vagus nerve with a single knot. It was used ethyl cyanoacrylate to adhere the vagus nerve to the silk suture, and surgical transection was completed.

NV elicited responses. The vagus nerve was placed on a moist tissue paper arranged on a custom acrylic platform and kept moist using artificial cerebrospinal fluid (126 mM NaCl, 26 mM NaHCO ₃ , 10 mM glucose, 2.5 mM KCl, ^{2 2} CaCl, 4 mM, 1.3 mM MgCh, and 1.2 mM NaH ² PO ⁴ ). A recording electrode (glass pipette filled with 2M NaCl) and a stimulating electrode (FHC, Bowdoin, ME) were placed over the vagus nerve, with the latter 4-5 mm away from the former (and further from the head). Signals were amplified (x1000, model 1800, AM Systems, Everett WA) and digitized (30 kHz) via an acquisition system (Micro1400 unit and Spike2 version 7 software, CED, Cambridge, UK). Stimuli (20 ps duration) of increasing intensity (1-50 V) were delivered with a stimulator (SD9, Grass, Warwick RI) to generate input/output curves (1 V increments in the range of 1-10, increments of 5 V in the range of 10-50), which were generated first in a saline solution and then in the environment of lidocaine (2%) or tetrodotoxin (100 μM). Responses were evaluated by integrating the area between the response curve and the baseline value (determined 1–10 ms before stimulation).

Data analysis. Spike2 software (version 7, CED) was used for analysis of raw recordings, which were filtered (using a high-pass filter with a 160 Hz edge) and smoothed. Neural signals were identified using user-specified adaptive threshold methodology. The identified compound action potentials (CAPs) were reviewed and signals erroneously captured by the adaptive threshold were manually removed. All areas of signal saturation, as well as signals corresponding to cardiac and respiratory components, were also ignored. Following this signal processing, information on velocity and temporal encoding patterns was extracted and further analyzed using OriginPro software (version 8, OriginLab, Northampton, MA). Neurograms were defined as the first time interval in which CAP frequency was >3x the baseline level. TNF and IL-1p neurograms were extracted and subjected to fast Fourier transform (FFT) for PSD analysis (3.9 Hz frequency resolution, using a Hanning window). In the frequency domain, notch filters (60 ± 10 and 120 ± 10 Hz) were applied to minimize the contribution of electrical noise along with its dominant harmonic. Data were linearly interpolated between notch-filtered intervals. Areas under the PSDs (range 20 to 400 Hz) were calculated for each cytokine response.

Statistical tests. Data are presented as individual samples, mean ± SD and mean ± SEM. The Shapiro-Wilk test was used to test for normality. ANOVA, Student's t test, Mann-Whitney U test, and Kolmogorov-Smirnov test were used to examine statistical significance. Values of p < 0.05 were considered significant.

Results

NV recordings capture neuronal compound action potentials. To investigate the neuronal activity of the vagus nerve, an electrophysiological recording system was constructed with three hook electrodes. Signals were digitized, subjected to high-pass filtering and post-acquisition smoothing algorithms (as described in Methods). The recorded signal showed periodic excursions from the reference situation that corresponded to cardiac (amplitude, 30-80 pV, with a duration of 4-5 ms) and respiratory (amplitude, ~50 pV, with a duration of ~120 ms) components. . Notably, contained within the signal were discrete aperiodic spikes of varying amplitude that lasted ∼2 ms ( Fig. 1A ). An adaptive threshold technique was used to identify these fast spikes (Fig. 1B) and assume that they corresponded to compound action potentials (CAPs), which were generated by the near-unison firing of multiple axons. To test whether the spikes truly reflected neural activity, nerve activation and inhibition studies were performed. In the first, after acquisition of the reference situation, KCl (4 mM) was applied to the surgical field very close to the vagus nerve to depolarize the neuronal tissue. This treatment produced a significant increase in neuronal activity (Fig. 1B), with individual CAPs showing large amplitude and a stereotypical shape (Fig. 1B). By electrically stimulating the vagus nerve preparation with brief pulses of increasing intensities, evoked CAPs of increasing amplitudes were obtained (Fig. 1C, D). The application of lidocaine, a potent anesthetic that blocks sodium channels, resulted in an almost complete disappearance of the evoked CAPs (Fig. 1C). On the other hand, application of tetrodotoxin, a highly specific sodium channel blocker, completely abrogated the evoked CAPs (Fig. 1D), corroborating the neuronal nature of the recordings. Notably, inhibition of neuronal activity did not completely eliminate the cardiac and respiratory components, which were likely a combination of electrocardiac signals, electromyographic signals, and motion artifacts (data not shown).

Afferent nature of NV neurograms. Examination of baseline vagus nerve activity indicated that unstimulated anesthetized mice showed minimal activation (Fig. 2A) with a mean underlying CAP frequency of 7.6 ± 1.6 Hz (mean ± SEM, N = 36 records with a duration >10 minutes). Traces were divided into 60-second intervals, which revealed long periods of complete quiescence (N = 85 events with CAP frequency = 0) that were interrupted by brief periods of strong neuronal activity. The CAP frequency distribution for non-silent events was not normally distributed (N = 275, P = 0, W = 0.69, Shapiro-Wilk test), with most events occurring at very low frequency and a few occurring in the high-frequency tail (Fig. 2B). An initial test of the hypothesis that the peripheral inflammatory state of an organism can represent a component of the sensory function of the vagus nerve by applying TNF (50 |jg in 200 jl, ip), while continuously recording the activity of the vagus nerve. The resulting postinjection neurogram showed an acute increase in neuronal activity (Fig. 2A), with a fraction of CAPs occurring at a much higher frequency than the baseline situation (Fig. 2B). The improved neurogram following TNF administration could reflect sensory signals, motor signals, or even both, traveling through the vagus nerve. To directly test the directionality of the signal, proximal and distal vagotomies (relative to the brain) were performed before administering TNF (Fig. 2C). With a proximal transection, the afferent fibers leading to the recording electrodes from the visceral organs remained intact, while the efferent fibers no longer interact with the electrodes. The opposite occurred with distal vagotomy, in which the recording electrodes no longer accessed the afferent fibers but the efferent fibers remained intact. For each experiment, CAPs were identified and mean CAP frequencies were plotted across the recordings (Fig. 2D). To evaluate responses, 10-minute intervals were considered immediately before and after TNF injection. With proximal vagotomy, CAP frequency showed a significant increase from baseline (pre, 5.8 ± 2.8; post, 39.5 ± 9.1 Hz, mean ± SEM). In marked contrast, distal vagotomy resulted in no improvement (pre, 5.7 ± 1.3; post, 2.9 ± 1.5 Hz, mean ± SEM). Comparison of individual postinjection CAP frequencies (Fig. 2E) further demonstrated the significant difference between proximal and distal transections (P = 0.03, T = 3.28, t test). These data indicate that the first response recorded in neurograms of the cervical vagus nerve, after cytokine administration, includes and requires the sensory function of the vagus nerve.

NV neurograms induced by proinflammatory atocins. To further dissect the neuronal activity associated with inflammatory stimuli, the patterns of responses between TNF and IL-1p were compared using purified exogenous proinflammatory cytokines that were injected into the peritoneal cavity. Saline injection was used as a control (Fig. 3A). Application of TNF (50 jg in 200 jl, ip) was followed by a notable increase in activity (Fig. 3B). Analysis of 10-minute intervals (immediately before and after TNF) revealed a significantly higher mean CAP frequency after TNF (pre, 4.1 ± 3.7, post, 19.8 ± 4.2 Hz, N = 6, P = 0.013, U = 2, Mann-Whitney test [MW]). The maximum CAP frequency, defined as the maximum value within the range, was also significantly elevated after TNF (pre, 19.8 ± 5.9, post, 69.0 ± 17.8 Hz, N = 6, P = 0.005, U = 0, MW test). Both the onset and termination of the activity curves were rapid, occurring on the order of seconds. Furthermore, the period following TNF-induced enhancement had a mean CAP frequency (7.4 ± 4.7 Hz) that was similar to baseline activity.

Vagus nerve neurograms were then assessed in the context of IL-1p injection (350 ng in 200 jl, ip). Interestingly, the maximal response to IL-1p occurred before that to TNF (Fig. 3C). For this reason, a 5-min interval was used to assess responses immediately before and after IL-1p injection. The mean frequency of ^{cA p} was significantly higher after cytokine (pre, 6.9 ± 3.6, post, 25.5 ± 4.9 Hz, mean ± SEM, N = 8, P = 0.007, U = 6, MW test), as well as the maximum CAP frequency (pre, 23.9 ± 11.3, post, 74 ± 10.2 Hz, N = 8, P = 0.013, U = 8, MW test) . After the IL-1p-enhanced neurogram, the mean CAP frequency returned to the baseline level (4.4 ± 1.9 Hz). Importantly, the increase in vagus nerve activity triggered by cytokines did not reflect physical trauma or alteration of the peritoneal cavity and its viscera because ip injection of vehicle alone (200 μl of sterile saline) did not trigger a change in vagus nerve activity (Fig. 3A), as demonstrated by mean CAP frequency (pre, 11.9 ± 4.9, post, 9.6 ± 4.1 Hz, N = 5, P = 1 , U = 12, MW test) and maximum frequency (pre, 106.6 ± 23.0, post, 46.0 ± 11.1 Hz, N = 5, P = 0.037, U = 23, MW test ).

To investigate the dose dependence of cytokine-induced neurogram improvement, the amount of proinflammatory cytokine administered was titrated. Reducing the amount of TNF tenfold (5 jg in 200 jl, i.p.) largely abolished the maximal response observed 5 min after injecting the highest dose (50 jg) of TNF (Fig. 3D). Similarly, CAP frequency did not increase at 150 seconds after IL-1p administration when it decreased tenfold (35 ng in 200 jl, ip) (Fig. 3E). The shorter time intervals used for IL-1p compared to TNF reflected the different temporal profiles of neurogram activation for IL-1p compared to TNF. Notably, the response to IL-1p peaked earlier (150 seconds after injection) compared to TNF (300 seconds after injection).

Differences between neurograms induced by TNF and IL-1p. Comparison of mean CAP frequencies (Fig. 4A), before and after treatment, revealed significant differences between the saline, TNF, and IL-1p groups (P = 0.0125, F[2, 21] = 5 .57, one-way ANOVA). Post-hoc tests also showed that the values of TNF and IL-1p were significantly different from those in saline (TNF, P = 0.006, U = 2; lL-1p, P = 0.01, U = 7, MW test). However, there was no difference between proinflammatory cytokines (P = 0.65, U = 20, MW test). Analysis of the maximum frequency of CAP (Fig. 4B), before and after treatment, showed statistical significance between the three groups (P = 6.5 x 10-4, F [2, 21] = 11.07, ANOVA). Post-hoc tests also demonstrated that maximal responses to TNF and IL-1p were significantly different from saline (TNF, P = 0.002, U = 0; IL-1p, P = 0.003, U = 3, MW test ). Additionally, CAP peak frequency changes among proinflammatory cytokines were not significantly different (P = 0.95, U = 23, MW test). The temporal profiles of neurogram activation with TNF and IL-1p treatments were compared by defining the onset of the response as the time at which the CAP frequency increased to 3x the preinjection rate, and the termination of the response as the time at which the frequency decreased below the same value. Using these definitions, CAP latency, duration, and mean frequency for responses were analyzed (Fig. 4C). Neurograms induced by TNF (N = 6) and IL-1p (N = 7) had different latencies (TNF, l56.2 ± 40.4, IL-1p, 47.6 ± 15.2 s, P = 0.043, T = 2.52, t test). In contrast, response duration (TNF, 185.2 ± 47.2, IL-1p, 222.6 ± 73.8 s, P = 0.68, T = 0.43, t test) and mean frequency of CAP (TNF, 35.92 ± 5.2, IL-ip, 42.7 ± 4.6 Hz, P = 0.35, T = 0.97, t test) were not different.

Receptor requirement for TNF- and IL-1p-induced neurograms. It has been previously shown that TNF trimers can form receptor-independent transmembrane channels (19). In this case, the administration of TNF could, in theory, directly activate neurons without involving its receptor. To test whether cytokine-induced responses required cytokine receptor signaling, neurograms were recorded from the vagus nerve of receptor knockout (KO) mice. Injection of TNF into TNFR2 KO mice did not lead to a change in the neurogram (Fig. 5A), implicating a ligand-receptor interaction in the observed response enhancement. Next, the requirement of the IL-1p receptor (IL-1R) in IL-1p-induced neurogram activation was tested. Exogenous IL-1p did not stimulate the cervical vagus nerve in IL-1R KO mice (Fig. 5B). In all experiments with KO mice in which no clear response was observed, KCl (4 mM) was applied to the nerve at the end of the experiments to ensure viability of the vagus nerve (data not shown). Additionally, in complementary crossover studies, ip administration of IL-1 in TNFR1/R2 double KO mice induced an increase in vagus nerve signaling (data not shown). Similarly, IL-1R KO mice responded to ip administration of TNF with increased vagus nerve activity (data not shown).

To test the requirement of full-length TNF for vagus nerve activation, recombinant human TNF was digested in 0.05% trypsin overnight at 37°C. Trypsin was heat inactivated by incubating the solution at 95 °C for 7 minutes. Approximately 90% digestion was confirmed by SDS-PAGE gel. Digested or full-length TNF (50 μg/animal) was injected into the peritoneum while continuously recording from the cervical vagus nerve. As expected, trypsin-digested TNF did not reproduce the effects of full-length TNF on vagus nerve activation (data not shown).

Spectral power of NV signals induced by TNF and IL-1p. Given that TNF and IL-1p trigger similar, but not identical, improvements in vagus nerve neurograms and that these cytokine receptor-dependent signals involved afferent fibers, we examined whether the vagus nerve is capable of sending distinct signals in relation to TNF. and IL-1p to the CNS. To investigate this possibility, power spectral densities (PSDs) of responses to TNF and IL-1p were analyzed using unfiltered neurogram recordings. As above, response was defined as the first time interval in which the CAP frequency resided at 3x the baseline level. Representative PSDs for TNF and IL-1p are shown in Fig. 6A. Each filtered PSD was then integrated (from 20 to 400 Hz) to compare individual areas of TNF and IL-1p responses (Fig. 6B). Statistical analysis revealed a significant difference between the TNF and IL-1p groups (N = 7, P = 0.05, D = 0.71, Kolmogorov-Smirnov test). Taken together, the difference in temporal characteristics of the response and frequency-domain power analyzes demonstrated selective neuronal sensory cytokine signaling through the vagus nerve.

Analysis

The work presented here adds to the rapidly expanding literature describing the interface between neuroscience and immunology, delineating for the first time the afferent arc of the inflammatory reflex. The use of surgical vagotomies verified the postulate that afferent fibers of the vagus nerve can function as cytokine sensors that transmit information to the CNS (5-7). Furthermore, neurogram analysis for TNF and IL-1p reveals specific and selective afferent signaling from the vagus nerve in response to respective proinflammatory cytokines.

Knockout (KO) mice were used to study the receptor dependence of the sensory component of the inflammatory reflex. The complete absence of neurogram improvement in KO animals for receptors paired with the administered cytokine (but not in KO animals for receptors mismatched with the administered cytokine) implicates the corresponding cytokine receptor in the specific cytokine-induced activation of the vagus nerve. . In summary, cytokine-receptor interactions mediate the neurogram response. At the cellular level, the mechanism by which systemic cytokines trigger neuronal activation in the vagus nerve remains an active area of research. Several studies have demonstrated the presence of functional cytokine receptors within neuronal populations, such that the activation of these receptors is capable of modulating neuronal excitability (10-12). A recent report has shown that bacterial products can directly activate a specific population of sensory neurons (13). It follows that proinflammatory cytokines can directly activate sensory vagus nerve fibers within the peritoneum. Alternatively, intermediate populations of receptor-expressing somatic cells may be required to detect the cytokine and, in turn, stimulate neurons.

Notably, the differences between TNF and IL-1p indicate that the CNS can discriminate between a diverse set of inflammatory mediators. This notion has a strong teleological basis because the CNS receives a continuous sensory flow pertaining to the internal environment of the body, from which the signals that transmit systemic inflammation They must constitute a key element for the homeostasis and survival of an animal. With rapid provision of signals about peripheral inflammatory (and inflammatory) status, an organism would be better able to initiate appropriate physiological and behavioral responses to immunological and environmental challenges.

In addition to the observed temporal differences in neurogram enhancement between TNF and IL-1p, the spectral densities of individual responses (Fig. 6) provide additional evidence for the existence of discriminatory features between cytokine-triggered vagus nerve activities that could be interpreted by CNS centers, such as the nucleus tractus solitarius. There is ample evidence that many cortical and subcortical structures within the CNS can distinguish the spectral features of signals and use them in processes as diverse as memory encoding, decision making, and switching between sleep states (14-16). . Either alone or in combination, the differences observed in time and frequency domain metrics may represent the biological substrate for peripheral cytokine discrimination by the sNc.

The interface between neuroscience, immunology and clinical medicine is increasingly coming to the fore, especially as the use of electrical devices as therapeutic agents for diseases becomes a reality and shapes the emerging field of bioelectronic medicine ( 17). For example, a recent clinical trial using vagus nerve stimulation to treat rheumatoid arthritis was successful (18).

EXAMPLE 2. Acoustic stimulation using IL1p-mediated neurogram induces increased levels of circulating inflammatory cytokines. Cytokine-related embodiments of Example 2 are not currently claimed, but are useful for understanding the invention.

The paradigm for inducing an inflammatory phenotype in naïve mice by acoustic stimulation using an IL1p-specific signal is illustrated in Fig. 7. Baseline and interleukin-1p (IL-1p)-specific neurograms were recorded from the vagus nerve. cervical of adult mice. The reference or IL1p-specific signal was extracted from the vagus nerve neurogram obtained from mouse A and converted to a 'Wav' file. The baseline or IL1p-specific signal was transferred to another naive recipient B mouse by acoustic stimulation ( ^1x or 3x serial activation). Circulating cytokine levels were analyzed in mouse B after 1 h. The IL1p-specific but not the baseline signal induced an inflammatory phenotype in naïve recipient mice (Fig. 8).

EXAMPLE 3. Neurograms of the vagus nerve specific for states of euglycemia, hyperglycemia and hypoglycemia.

Hypoglycemic neurogram recording. Naïve mice received a bolus injection of insulin (6 mg/kg) and vagus nerve activity was recorded over time (Fig. 9). Blood glucose levels were monitored after every 2.5 minutes. Insulin induced a hypoglycemic state in mice. Blood glucose levels are indicated on the top line of Fig. 11 in mg/dl.

Euglycemic neurogram recording. Naïve mice received a bolus injection of glucagon (1 mg/kg) and vagus nerve activity was recorded over time (Fig. 10). Blood glucose levels were monitored every 2.5 minutes. Blood glucose levels are indicated on the top line of Fig. 11 in mg/dl.

Hyperglycemic neurogram recording. Naïve mice received a bolus injection of glucose, and vagus nerve activity was recorded over time (Fig. 11). Blood glucose levels were monitored every 2.5 minutes. Glucose induced a hyperglycemic state in mice. Blood glucose levels are indicated on the top line of Fig. 13 in mg/dl.

EXAMPLE 4. Control of blood glucose levels in human subjects through acoustic reproduction of vagal neurograms specific for states of hyperglycemia or hypoglycemia

Effects on blood glucose levels in healthy human subjects were determined in response to acoustic playback of hypoglycemia-specific neurograms, hyperglycemia-specific neurograms, or euglycemia-specific neurograms.

Fig. 12 illustrates a paradigm for increasing blood glucose levels in healthy human subjects by acoustic stimulation using a hypoglycemia-specific signal recorded from the vagus nerve. Naïve mice received a bolus injection of insulin (6 mg/kg) and vagus nerve activity was recorded over time. Insulin induced a hypoglycemic state in mice. The signal was converted into a 'wav' file and transferred as audio output to healthy human subjects. Blood glucose levels were monitored in human subjects at regular time intervals. Acoustic playback of the hypoglycemia-specific neurogram induced hyperglycemia in healthy human subjects (Fig. 13).

In another set of experiments, vagus nerve neurograms were recorded from naïve mice that received a bolus injection of glucose, and vagus nerve activity was recorded over time. Blood glucose levels increased in the mice given glucose. The neurogram of the vagus nerve was converted into a 'wav' file and transferred as audio output to healthy human subjects. Blood glucose levels were monitored over time (n=3/group). Acoustic playback of the hyperglycemia-specific neurogram induced hypoglycemia in healthy human subjects (Fig. 14).

In a set of control experiments, vagus nerve neurograms were recorded from naïve mice that received a bolus injection of glucagon (1 mg/kg), and vagus nerve activity was recorded over time. Blood glucose levels did not change in mice given glucagon. The neurogram was then played back as a 'wav' file for healthy human subjects and blood glucose levels were monitored over time (n=5/group). Acoustic playback of the euglycemia-specific neurogram did not induce a change in blood glucose levels in healthy human subjects (Fig. 15).

EXAMPLE 5. Acoustic playback in human subjects using cortisol-specific vagal neurogram induces hyperglycemia

Mouse hydrocortisone-induced neurogram recording. Injection of one bolus of hydrocortisone (cortisol) (10 mg/mouse) into naïve mice induced a specific neurogram that was recorded from the vagus nerve (Fig. 16).

Cortisol administration induces hypoglycemia in mice. Naïve mice received saline or cortisol via intraperitoneal administration. Blood glucose levels were monitored over time. Cortisol administration, but not saline administration, induced hypoglycemia (Fig. 17).

Acoustic playback of the cortisol-specific neurogram induced hyperglycemia in healthy human subjects. A cortisol-specific signal was extracted from a neurogram obtained from the vagus nerve of a mouse after ip administration of cortisol. To generate a control signal, the cortisol-specific signal was transformed into a scrambled signal using a matlab code that generates a randomized signal with the same amplitudes, but random frequencies, maintaining the full power of the signal. The cortisol-specific or scrambled signal was converted into a 'wav' file and transferred as audio output to healthy human subjects. Blood glucose levels were monitored in human subjects at regular time intervals. Acoustic playback of the cortisol-specific neurogram induced hyperglycemia in healthy human subjects (Fig. 18).

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Claims (3)

1. A method of producing a .wav file wherein the .wav file can be played as an acoustic energy stimulus for subsequent use in the treatment of a subject having a disease or disorder, wherein the disease or disorder is hyperglycemia or hypoglycemia, the method comprising generating the .wav file based on a neurogram of the vagus nerve specific to an endogenous mediator or specific to a pharmacological agent, the method comprising extracting a portion of the neurogram previously recorded from a vagus nerve and converting it into the ".wav" file, where - the acoustic energy stimulus is for subsequent use to increase blood glucose levels in the subject wherein the neurogram is an insulin-specific or cortisol-specific neurogram that has been recorded from the vagus nerve after administration of insulin or cortisol, or - the acoustic energy stimulus is for subsequent use to lower blood glucose levels in a subject wherein the neurogram is a glucose-specific neurogram that has been recorded from the vagus nerve after glucose administration. 2. The method of claim 1, wherein the neurogram specific for an endogenous mediator or specific for a pharmacological agent is obtained using an animal model or from a human patient. 3. The method of claim 1, wherein the neurogram specific for an endogenous mediator or specific for a pharmacological agent is one that has been obtained from a branch of the vagus nerve to a specific organ or portion of an organ.